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,1
,
* Department of Human Genome Technology, Kausa DNA Research Institute;
Laboratory of Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Chiba University, Kisarazu, Chiba, Japan;
Laboratory for Developmental Genetics, Research Center for Allergy and Immunology, Institute of Physical and Chemical Research (RIKEN), Yokohama, Kanagawa, Japan; and
Laboratory for Immunogenomics, Research Center for Allergy and Immunology, Institute of Physical and Chemical Research (RIKEN), Yokohama, Kanagawa, Japan
1Correspondence: 2–6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818 Japan. E-mail: nmanabu{at}kazusa.or.jp
SPECIFIC AIMS
Given that thousands of genes exist in the mammalian genome, criteria are needed to prioritize their functional analysis and to decrease the likelihood of producing gene-targeted mice that lack overt phenotypes. The aim of this study is to demonstrate that initial analysis efforts are likely to be fruitful if focused on genes encoding large proteins, since at least some large proteins serve as frameworks for intricate assembly of protein complexes and their inactivation would render definitive, observable phenotypes (Fig. 1
).
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PRINCIPAL FINDINGS
1. Comprehensive gene analyses using gene-targeted mice requires a systematic, high-throughput approach
Because constructing gene-targeted mice is time-consuming, laborious, and costly, we have implemented an economical and systematic approach for constructing our five KIAA gene-targeted mice. First, the human KIAA genes analyzed in the present study were selected according to three criteria, size, protein homology, and tissue-specific expression, that would increase the probability that their disruption would produce an observable, measurable phenotype. Thus, we chose genes encoding proteins with 1000 amino acids or more, having <35% shared homology with known proteins, encoding proteins with low expectation values (to avoid compensational effects), and those being expressed primarily in the brain (neural defects are readily manifested phenotypically). We especially focus on the function of proteins having no homology with known proteins and few known motifs. Because the function of such proteins cannot be predicted in silico, we strongly believe that experimental analysis of such proteins should be performed with higher priority to have overall knowledge about the organism.
Second, after identifying the target genes, we sought a rapid means to screen BAC clones containing genomic DNA fragments of mouse homologue KIAA genes and to increase the probability of their homologous recombination into the genome of mouse embryonic stem (ES) cells. Efficient screening of BAC clones containing mouse homologue KIAA genes was carried out using a polymerase chain reaction-based BAC library screening system that consisted of 
40,000 clones, the aligned sequences of which were plated onto 384-well plates. The clones were derived from chromosomal DNA extracted from R1 ES cells, the same cell type used for homologous recombination and implantation into pseudopregnant female mice. We made our vectors using the identical genomic DNA of recipient R1 ES cells, because the frequency of homologous recombinant increases when the targeting vector is constructed with DNA derived from recipient ES cells. We attained a high frequency of homologous recombination: 1 in 9, 1 in 4, 1 in 7, 1 in 9, and 1 in 27 targeting vectors containing knockouts of KIAA1409, KIAA1440, KIAA1447, KIAA1768, and KIAA1276, respectively, successfully recombined.
2. Viability of KIAA gene-targeted mice
To examine the viability of homozygotes of the five sets of KIAA gene-targeted mice, we mated heterozygotes, genotyped their pups at various stages of preand postnatal development, and compared the homozygotes to their heterozygote littermates. Of the five sets of mice, two were fully viable (KIAA1276 and KIAA1768) and showed no overt phenotypes. The remaining three (KIAA1409, KIAA1440, and KIAA1447) died during embryonic development or shortly after birth. Only one KIAA1447 homozygote mouse grew to adulthood. Thus three of five sets of gene-targeted mice showed clear congenital defects during development, indicating that these three KIAA genes play an essential role in normal development.
3. KIAA1409–/– mice lack the ability to drink milk and die within few days
Although the large majority of KIAA1409–/– homozygote mice died at (P0) or the day after (P1) birth (Fig. 2
), a few mice survived to P7. As shown in Fig. 2C
, homozygote and heterozygote KIAA1409 gene-targeted mice differed greatly in size and appearance. We observed milk within the stomachs of heterozygote and wild-type (WT) pups but not in the stomachs of homozygote pups. Although newborn homozygote pups were active soon after birth, after half a day, the pups appeared weak and their movements decreased gradually, presumably due to their inability to consume milk (Fig. 2C
). Over 90% of KIAA1409–/– pups failed to nurse. Almost no milk was found in the stomachs of dead and live homozygotes examined at P0. The remaining 10% drank very little milk in comparison to KIAA1409+/– and KIAA1409+/+ pups, the latter of which had full stomachs of milk (Fig. 2C
). Thus, unlike their heterozygote and WT littermates, KIAA1409–/–pups surviving 2–4 days after birth gained virtually no weight. KIAA1409–/– pups surviving to P6 or P7 were very thin, and their gradual weakening and ensuing death are most likely due to their inability to nurse. Placing the KIAA1409–/– pups in the care of either WT or ICR mothers did not improve this situation.
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4. Disruption of KIAA1440 led to embryonic lethality at the blastocyst stage
Unlike the KIAA1409–/– homozygotes, no viable KIAA1440–/– homozygotes were born (Fig. 3
A). To determine at which developmental stage these mice died, we attempted to examine their viability at E9.5, E11.5, and E13.5 of gestation, but none could be identified at these time points (Fig. 3A
). In addition, we were unable to find resorbed embryos, suggesting KIAA1440–/– embryos die very early during development, perhaps before implantation. Examination of preimplantation embryos isolated and genotyped at E3.5 revealed that KIAA1440–/– blastocysts were morphologically distinguishable from heterozygote and WT blastocysts. Thus, we concluded KIAA1440–/– embryos stop developing at the morula stage (Fig. 3B
).
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5. Adult KIAA1447–/– mouse had overt motor deficits
The majority of KIAA1477–/– homozygote mice died shortly after birth, indicating that KIAA1477 protein contributes to postnatal development of mice. Despite the high mortality of KIAA1477–/– neonates, we were able to obtain one adult KIAA1447–/– mouse. This mouse exhibited a markedly obvious phenotype. Control heterozygote mice move about effortlessly. The KIAA1447–/– mouse, however, had great difficulty walking, often dragging its hind legs. Unlike its heterozygote littermates, the hindquarters of the KIAA1447–/– mouse were positioned abnormally, such that its loins sagged and its abdomen touched the floor. Quantitative assessment of motor activity in an open field test, as well as subsequent assessment of spontaneous locomotor activity, revealed significant motor/exploratory differences between KIAA1447 heterozygote and homozygote mice.
CONCLUSIONS AND SIGNIFICANCE
To make progress in diagnosing and treating inherited diseases, it is necessary to identify and characterize the genes responsible for such diseases. In addition to loci mapping and disease association study, targeted mutations and gene knockouts eliciting overt phenotypic alterations provide powerful tools for studying gene function and identifying candidate genes involved in disease. A catalog of gene-targeted mice, cross-referenced according to the gene disrupted and the resulting phenotype, will greatly aid the ongoing search for genes responsible for human inherited diseases. At present, the difference between a gene that expresses a clear phenotype and a gene that fails to express a clear phenotype is unknown. When a gene is disrupted and subsequently expressed in a mouse, the gene-targeted mouse often appears outwardly normal. To avoid wasting time in constructing such mice, it is very important to be aware of which genes, even when disrupted, express normal-looking phenotypes. There are numerous potential reasons for the lack of discernible phenotypic alterations in gene-targeted mice. With respect to the points concerning gene compensation and hidden or minute phenotypic alterations, because large proteins possess specialized functions, compensation by other proteins is unlikely. Hidden or minute phenotypic alterations are also unlikely because the disruption of a large protein would affect the overall function of protein complexes comprised of many proteins. For these reasons, we hypothesize that the disruption of a gene encoding a large protein results in obvious phenotypic alterations in a gene-targeted mouse because large proteins play a central role in the function of protein complexes.
Because future analyses using gene-targeted mice will likely use uncharacterized genes, the risk will increase significantly that gene-targeted mice lacking distinct phenotypic alterations will be obtained. By using our gene selection criteria, however, a researcher can lower this risk by avoiding most factors that contribute to lack of discernible phenotypes in these mice. Since our success rate is sufficiently high, it would be worthwhile to use despite the possibility of failure for the aforementioned reasons.
In summary, we used a systematic, rapid, and efficient approach to assess the function of genes that encode large proteins in transgenic knockout mice. Of the five different sets of KIAA gene-targeted mice constructed, the three harboring knockouts of the largest genes showed distinct phenotypes, indicating that KIAA1409, KIAA1440, and KIAA1447 are essential to normal development. Moreover, our approach brings a new perspective to the world of biology: a protein complex can be thought of as being organized hierarchically in terms of function. Some proteins are essential for the proper functioning of and integrity of the complex; others are relatively less important, taking on modifier roles. We propose that extremely large proteins are the key element of a protein complex because they function as the framework around which the complex is assembled. In this study, we made five gene-targeted mice, each harboring one of five KIAA genes encoding proteins 2596 aa, 2222 aa, 2644 aa, 1742 aa, or 942 aa in length. The gene-targeted mice with disruptions of the three largest proteins exhibited clear phenotypes, but mice with disruptions of the relatively smaller KIAA proteins failed to exhibit observable phenotypes. This result is in agreement with the hypothesis that we are advocating. This report represents the first comprehensive, functional assessment of proteins of unknown function derived from a cDNA sequencing project. We will continue our experiments with the goal of increasing the number of phenotypically verifiable KIAA knockout mice in line with our hypothesis that large proteins with multiple binding domains play central roles in functional protein complexes, the disruption of which results in clear phenotypic manifestations.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5952fje
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